Chromatin from cultured cells, mouse PVN punches (individual pools formed from groups of 3) or whole hypothalami dissected from fresh brains were cross linked, disrupted by sonification and purified.

The binding of Suz12 to wingless (Wnt1), a beta-catenin dependent developmental regulator and to the RNA polymerase II promoter (RNAPII), a housekeeping gene, served as positive and negative controls, respectively, in these experiments.

Chromatin was prepared from U-2 OS (Human bone osteosarcoma epithelial cell line) cells according to the Abcam X-ChIP protocol.

Cells were fixed with formaldehyde for 10 minutes. The  ChIP was performed with 25 µg of chromatin, 2 µg of  ab5131 (blue), and 20 µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified on the inactive AFM and F8 promoters, the GAPDH promoter (active) and over the y-Actin gene (active).

Schematic diagram of the y-Actin gene is shown on the top of the figure. Black boxes represent exons and thin lines represent introns. PCR products are depicted as bars under the gene.

All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody (ab5131) at 1/5000 dilution

Lane 1 : Xenopus laevis whole tissue lysate treated with DMSO for 24 hours
Lane 2 : Xenopus laevis whole tissue lysate treated with CDK inhibitor for 24 hours

Secondary
All lanes : HRP-conjugated goat anti-rabbit IgG polyclonal at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 217 kDa
Observed band size: 240 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute

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HeLa (Human epithelial cell line from cervix adenocarcinoma) cells were fixed with 4% formaldehyde in PEM buffer.

Sample was incubated in blocking buffer of 5% powdered milk in TBS-T plus 0.02% sodium azide for 1 hour at room temperature. Blocking buffer was removed and primary antibody was added at a dilution of 1/160 and incubated overnight at 4 degrees celsius. Sample then washed 4-5 times with blocking buffer for 5 minutes. Secondary antibody, goat anti-rabbit Alexa ^®594, was added at a dilution of 1/1000 and incubated at room temperature for one hour.

From this point on samples were covered with foil to protect them from light. They were washed 5 times with TBS-T and then one time with PEM, for 5 minutes each wash; fixed 10-30 minutes in 4% formaldehyde in PEM buffer, then washed 3 times with PEM buffer for 5 minutes. 0.1M ammonium chloride in PEM buffer was added for 10 minutes to quench auto-florescence, and then slips were washed 2 times for 5 minutes in PEM followed by 3 washes for 5 minutes.

ICC/IF image of ab5131 stained HeLa(Human epithelial cell line from cervix adenocarcinoma) cells.

The cells were fixed in methanol for 5 minutes, permabilized in TBS-T  for 20 minutes and incubated with the antibody (ab5131, 1 µg/ml) for 1 hour at room temperature. 1% BSA / 10% normal goat serum / 0.3M glycine was used to quench auto-fluorescence and block non-specific protein-protein interactions.

The secondary antibody (green) was Alexa Fluor^® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1 hour. Alexa Fluor^® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).

All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody (ab5131) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate
Lane 2 : S.cerevisiae (Y190) Whole Cell Lysate
Lane 3 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate with S. cerevisiae RNA polymerase II CTD repeat YSPTSPS peptide (ab12795) at 1 µg/ml
Lane 4 : S.cerevisiae (Y190) Whole Cell Lysate with S. cerevisiae RNA polymerase II CTD repeat YSPTSPS peptide (ab12795) at 1 µg/ml
Lane 5 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate with Human RNA polymerase II CTD repeat YSPTSPS (phospho S5) peptide (ab18488) at 1 µg/ml
Lane 6 : S.cerevisiae (Y190) Whole Cell Lysate with Human RNA polymerase II CTD repeat YSPTSPS (phospho S5) peptide (ab18488) at 1 µg/ml
Lane 7 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate with S. cerevisiae RNA polymerase II CTD repeat YSPTSPS (phospho S1606 + S1613) peptide (ab12793) at 1 µg/ml
Lane 8 : S.cerevisiae (Y190) Whole Cell Lysate with S. cerevisiae RNA polymerase II CTD repeat YSPTSPS (phospho S1606 + S1613) peptide (ab12793) at 1 µg/ml

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 217 kDa


Exposure time: 30 seconds

Lanes 1 & 3-4 : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody (ab5131) at 1/500 dilution
Lane 2 : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody (ab5131) at 1/2000 dilution

Lanes 1-2 : HeLa nuclear extract
Lane 3 : HeLa nuclear extract with Human RNA polymerase II CTD repeat YSPTSPS (phospho S5) peptide (ab18488) at 1 µg/ml
Lane 4 : HeLa nuclear extract with S. cerevisiae RNA polymerase II CTD repeat YSPTSPS peptide (ab12795) at 1 µg/ml

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution

Performed under reducing conditions.

Predicted band size: 217 kDa
Observed band size: 250 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds